Search

Multi-drug resistance P-glycoprotein (P-gp/MDR1) is one of the most clinically relevant ABC transporters, highly enriched at the blood-brain barrier (BBB) with a broad substrate spectrum including therapeutic drugs and metabolic waste products. Altered P-gp transport function has been implicated in multi-drug resistance and in the pathogenesis and progression of neurological diseases. Recent studies have shown that P-gp expression is modulated by micro-RNAs in peripheral organs. Particularly, miR-27a-3p has been shown to play a critical role in the regulation of P-gp in multi-drug resistant cancer cells. In brain disorders, altered levels of miR-27a-3p were reported in several diseases associated with alterations in P-gp expression at the BBB. However, effect of altered miR-27a-3p expression on P-gp expression at the BBB remains to be determined. In this study, we investigated the role of miR-27a-3p in the regulation of P-gp expression and activity at the brain endothelium. Levels of miR-27a-3p were modulated by mimic and inhibitor transfection in an in-vitro model of human brain endothelial hCMEC/D3 cells. Effect of miR-27a-3p modulation on P-gp expression and activity was examined and the underlying regulatory mechanisms explored. Our results showed that transfection of hCMEC/D3 cells with miR-27a-3p mimic induces expression and activity of P-gp while miR-27a-3p inhibition exerted opposite effects. Mechanistic studies revealed that miR-27a-3p regulates P-gp by mediating Glycogen Synthase Kinase 3 Beta (GSK3s) inhibition and activating Wnt/s-catenin signaling. These findings shed light on miR-27a-3p/GSK3s/s-catenin as a novel axis that could be exploited to modulate P-gp efflux activity at the brain endothelium and help improving CNS diseases treatment or brain protection.

Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 +/- 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa. Funding Agencies|European TRANS-INT Consortium; European Union [281035]; TRANS-INT; Swedish Research Council [2822, 2020-01586]; FRS-FNRS (Fonds de la Recherche Scientifique) , Belgium; Competitive Reference Groups, Conselleria de Educacion e Ordenacion Universitaria, Xunta de Galicia [ED431C 2021/17]; European Commission, Education, Audiovisual and Culture Executive Agency (EACEA) [20120028]; Swedish Government, Sweden

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells.Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE.We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA.These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

BackgroundBrain metastasis (BM) is a major cause of morbidity and mortality in breast cancer (BC) and its molecular mechanism remains poorly understood. Transmigration of metastatic cells through the brain endothelium is an essential step in BM. Metalloproteinase-1 (MMP-1) overexpression plays a key role in promoting trans-endothelial migration by degrading the inter-endothelial junctions and disrupting the endothelial integrity. However, little is known about the molecular mechanisms that induce MMP-1 in metastatic cells granting them a brain invasive phenotype. MiR-202-3p is downregulated in brain metastases compared to primary breast tumors and directly targets MMP-1. Here, we unraveled a critical role of miR-202-3p loss in MMP-1 upregulation promoting transmigration of metastatic cells through the brain endothelium.MethodsA variant of the MDA-MB-231 human BC cell line (MDA-MB-231-BrM2) selected for its propensity to form brain metastases was found to express high levels of MMP-1 and low levels of miR-202-3p compared to the parental cells. Using a gain-and-loss of function approach, we modulated levels of miR-202-3p and examined the resultant effect on MMP-1 expression. Effect of miR-202-3p modulation on integrity of the brain endothelium and the transmigrative ability of BC cells were also examined.ResultsLoss of miR-202-3p in breast cancer cells enhanced their transmigration through the brain endothelium by upregulating MMP-1 and disrupting the inter-endothelial junctions (claudin-5, ZO-1 and ß-catenin). Restoring miR-202-3p exerted a metastasis-suppressive effect and preserved the endothelial barrier integrity.ConclusionsOur study identified a critical regulatory role of miR-202-3p in brain metastasis and shed light on miR-202-3p/MMP-1 axis as a novel prognostic and therapeutic target that can be exploited to predict and prevent brain metastasis in breast cancer patients.

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ET(A) and ET(B)) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ET(B) receptors (hET(B)) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ET(B) receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ET(A) receptor (hET(A)). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hET(B) receptor only some of new antibodies directed against ET(A) receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hET(A) is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

The objective of this work was the development of a new drug nanocarrier intended to overcome the barriers associated to the oral modality of administration and to assess its value for the systemic or local delivery of peptides. The nanocarrier was rationally designed taking into account the nature of the intestinal barriers and was loaded with insulin, which was selected as a model peptide. The nanocarrier consisted of a complex between insulin and a hydrophobically-modified cell penetrating peptide (CPP), enveloped by a protecting polymer. The selected CPP was octaarginine (r8), chemically conjugated with cholesterol (Chol) or lauric acid (C12), whereas the protecting polymer was poly (glutamic acid)-poly (ethylene glycol) (PGA-PEG). This enveloping material was intended to preserve the stability of the nanocomplex in the intestinal medium and facilitate its diffusion across the intestinal mucus. The enveloped nanocomplexes (ENCPs) exhibited a number of key features, namely (i) a unimodal size distribution with a mean size of 200 nm and a neutral zeta potential, (ii) the capacity to associate insulin (~100% association efficiency) and protect it from degradation in simulated intestinal fluids, (iii) the ability to diffuse through intestinal mucus and, most importantly, (iv) the capacity to interact with the Caco-2 model epithelium, resulting in a massive insulin cell uptake (47.59 ± 5.79%). This enhanced accumulation of insulin at the epithelial level was not translated into an enhanced insulin transport. In fact, only 2% of insulin was transported across the monolayer, and this was correlated with a moderate response of insulin following oral administration to healthy rats. Despite of this, the accumulation of the insulin-loaded nanocarriers in the intestinal mucosa could be verified in vivo upon their labeling with 99m Tc. Overall, these data underline the capacity of the nanocarriers to overcome substantial barriers associated to the oral modality of administration and to facilitate the accumulation of the associated peptide at the intestinal level.

International audience; Notwithstanding potential neurotoxicity of inhaled titanium dioxide nanoparticles (TiO 2 NPs), the toxicokinetics and consequences on blood-brain barrier (BBB) function remain poorly characterized. To improve risk assessment, we need to evaluate the impact on BBB under realistic environmental conditions and take into account vulnerability status such as age. 12–13 week and 19-month-old male rats were exposed by inhalation to 10 mg/m 3 of TiO 2 nano-aerosol (6 hrs/day, 5 day/week, for 4 weeks). We showed an age-dependent modulation of BBB integrity parameters suggesting increased BBB permeability in aging rats. This alteration was associated with a significant increase of cytokines/ chemokines in the brain, including interleukin-1β, interferon-γ, and fractalkine as well as a decreased expression of synaptophysin, a neuronal activity marker. These observations, in absence of detectable titanium in the brain suggest that CNS-related effects are mediated by systemic-pathway. Moreover, observations in terms of BBB permeability and brain inflammation underline age susceptibility. Even if TiO 2 NPs were not evidenced in the brain, we observed an association between the exposure to TiO 2 NPs and the dysregulation of BBB physiology associated with neuroinflammation and decreased expression of neuronal activity marker, which was further exacerbated in the brain of aged animal's. Due to their remarkable properties, nanoparticles (NPs) could potentially be used in a large array of applications , from electronics to medicine. Nanomaterials are currently being used in the production or manufacturing processes of a number of commercial products 1. In this context, there are increasing concerns regarding the potential adverse effects of NPs on human health. Among the wide variety of nanomaterials, titanium dioxide NPs (TiO 2 NPs) are produced in a large industrial scale and can be found in commercial products such as paints, food additives 2 , cosmetics 3 , and environmental decontamination systems 4. Published data from studies in rodents show that TiO 2 NPs can cross the epithelial barriers (nasal, bronchial, alveolar, gastro-intestinal) and enter the blood circulation from the respiratory and the gastro-intestinal tracts 5–10. After inhalation exposure , there is larger concern about translocation of NPs into the brain, either directly via the olfactory pathway or indirectly across the blood brain barrier (BBB). Indeed, ambient air particles and NPs have been previously demonstrated to translocate into the brain after inhalation, and thus may potentially influence the central nervous system (CNS). For example, translocation of 36 nm 13 C particles was observed in the olfactory bulb of rats, most likely originating from entry via the olfactory mucosa of the nose 11. Brain translocation of gold NPs were also observed in a rat exposure model 12 , and 15–20 nm iridium NPs could be detected in the rat brain up to 6 months after inhalation exposure 13,14. Despite these observations, the effects of NPs on the CNS under realistic; Malgré les études suggérant une neurotoxicité potentielle des nanoparticules de dioxyde de titane (NPs TiO2), peu de données portent sur la toxico-cinétique de ces nanoparticules et sur leur impact sur la barrière hémato-encéphalique (BHE). Afin d’améliorer cette évaluation du risque, il est nécessaire d’étudier l’impact des NPs TiO2 sur la BHE dans des conditions d’exposition réalistes et de prendre en considération un paramètre important qui est la vulnérabilité liée à l’âge. Des rats âgés de 12 à 13 semaines et de 19 mois ont été exposés par inhalation à un nano-aérosol de TiO2 à la concentration de 10 mg/m3, 6 heures/jour, 5 jours/semaine pendant 4 semaines. Il a été montré, une modulation des paramètres d’intégrité de la BHE en fonction de l’âge de l’animal. En effet, chez les rats âgés, il a été constaté une augmentation de la perméabilité de la BHE. Cette altération est associée à une augmentation significative des cytokines/chimiokines dans le cerveau (interleukine 1 b, interféron g, et fractalkine) mais également à une diminution de l’expression de la synaptophysine, marqueur de l’activité neuronale. En l’absence de titane détectable dans le cerveau, ces données suggèrent que les effets observés au niveau du système nerveux central sont transmis par voie systémique. Malgré l’absence de translocation cérébrale, un lien existe entre l’exposition aux NPs TIO2 et au dysfonctionnement de la BHE associé à une neuro-inflammation et une diminution de l’expression de l’activité neuronale qui est exacerbée chez les rats âgés.

The aim of this work was to rationally design and characterize nanocapsules (NCs) composed of an oily core and a polyarginine (PARG) shell, intended for oral peptide delivery. The cationic polyaminoacid, PARG, and the oily core components were selected based on their penetration enhancing properties. Insulin was adopted as a model peptide to assess the performance of the NCs. After screening numerous formulation variables, including different oils and surfactants, we defined a composition consisting of oleic acid, sodium deoxycholate (SDC) and Span 80. This selected NCs composition, produced by the solvent displacement technique, exhibited the following key features: (i) an average size of 180 nm and a low polydispersity (0.1), (ii) a high insulin association efficacy (80–90% AE), (iii) a good colloidal stability upon incubation in simulated intestinal fluids (SIF, FaSSIF-V2, FeSSIF-V2), and (iv) the capacity to control the release of the associated insulin for > 4 h. Furthermore, using the Caco-2 model cell line, PARG nanocapsules were able to interact with the enterocytes, and reversibly modify the TEER of the monolayer. Both cell adhesion and membrane permeabilization could account for the pronounced transport of the NCs-associated insulin (3.54%). This improved interaction was also visualized by confocal fluorescent microscopy following oral administration of PARG nanocapsulesto mice. Finally, in vivo efficacy studies performed in normoglycemic rats showed a significant decrease in their plasma glucose levels after treatment. In conclusion, here we disclose key formulation elements for making possible the oral administration of peptides This work was supported by the European TRANS-INT Consortium, which received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 281035. Z. Niu also would like to thank the Chinese Scholarship Council for his scholarship SI

The creatine transporter deficiency is a neurological disease caused by impairment of the creatine transporter SLC6A8, resulting in mental retardation associated with a complete absence of creatine within the brain and cellular energy perturbation of neuronal cells. One of the therapeutic hypotheses was to administer lipophilic creatine derivatives which are (1) thought to have better permeability through the cell membrane and (2) would not rely on the activity of SLC6A8 to penetrate the brain. Here, we synthesized creatine fatty esters through original organic chemistry process. A screening on an in vitro rat primary cell-based blood-brain barrier model and on a rat primary neuronal cells model demonstrated interesting properties of these prodrugs to incorporate into endothelial, astroglial, and neuronal cells according to a structure-activity relationship. Dodecyl creatine ester showed then a 20-fold increase in creatine content in pathological human fibroblasts compared with the endogenous creatine content, stating that it could be a promising drug candidate.

Proteolytic cleavage of the amyloid-beta protein precursor (A beta PP) ecretases leads to extracellular release of amyloid-beta (A beta) peptides. Increased production of A beta(42) over A beta(40) and aggregation into oligomers and plaques constitute an Alzheimer's disease (AD) hallmark. Identifying products of the 'human chemical exposome' (HCE) able to induce A beta(42) production may be a key to understanding some of the initiating causes of AD and to generate non-genetic, chemically-induced AD animal models. A cell model was used to screen HCE libraries for A beta(42) inducers. Out of 3500+ compounds, six triazine herbicides were found that induced a beta- and gamma-secretases-dependent, 2-10 fold increase in the production of extracellular A beta(42) in various cell lines, primary neuronal cells, and neurons differentiated from human-induced pluripotent stem cells (iPSCs). Immunoprecipitation/mass spectrometry analyses show enhanced production of A beta peptides cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and lower, a characteristic of AD. Neurons derived from iPSCs obtained from a familialAD(FAD) patient (A beta PP K724N) produced more A beta(42) versus A beta(40) than neurons derived from healthy controls iPSCs (A beta PP WT). Triazines enhanced A beta(42) production in both control and AD iPSCs-derived neurons. Triazines also shifted the cleavage pattern of alcadein alpha, another gamma-secretase substrate, suggesting a direct effect of triazines on gamma-secretase activity. In conclusion, several widely used triazines enhance the production of toxic, aggregation prone A beta(42)/A beta(43) amyloids, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in late-onset AD.

International audience; Our current knowledge about drug transporters in the maturational brain is very limited. In this study, we provide a comprehensive overview of the expression and activity profile of P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters during blood-brain barrier (BBB) maturation. Gene and protein expressions of the analyzed transporters increase as the brain matures, with no variation in their activity for P-gp and bcrp, while the transport activity of oat3 and oatpla4 increases during brain maturation from preterm up to adulthood. For the first time, we illustrate a downregulation of nuclear beta-catenin expression in brain capillaries when bcrp, P-gp, oat3, and oatpla4 transporters are at their highest expression levels. In vivo activation of beta-catenin in rat brains, by intracerebroventricular (ICV) injection of a GSK-3 inhibitor, enhances the activity of P-gp, bcrp, oat3, and oatp1a4. Interestingly, in an in vitro BBB model consisting of a coculture of primary endothelial brain cells with astrocytes or in vivo, activation of beta-catenin enhances the mRNA expression of ET -1. Interestingly, blocking the ETA receptor for endothelin-1 in vivo by ICV injection of a ETA antagonist decreases transporter activity mediated by the activation of beta-catenin. These findings shed light on the role of an interaction between beta-catenin and endothelin-1 signaling in the regulation of these transporters at the BBB.

We have evaluated a novel in vitro cell-based human blood-brain barrier (BBB) model that could predict in vivo human brain penetration for compounds with different BBB permeabilities using the clinical positron emission tomography (PET) data. Comparison studies were also performed to demonstrate that the in vitro cell-based human BBB model resulted in better predictivity over the traditional permeability model in discovery organizations, Caco-2 cells. We evaluated the in vivo BBB permeability of [(18)F] and [(11)C]-compounds in humans by PET imaging. The in vivo plasma-brain exchange parameters used for comparison were determined in humans by PET using a kinetic analysis of the radiotracer binding. For each radiotracer, the parameters were determined by fitting the brain kinetics of the radiotracer using a two-tissue compartment model of the ligand-receptor interaction. Bidirectional transport studies with the same compounds as in in vivo studies were carried out using the in vitro cell-based human BBB model as well as Caco-2 cells. The in vitro cell-based human BBB model has important features of the BBB in vivo and is suitable for discriminating between CNS and non-CNS marketed drugs. A very good correlation (r(2) = 0.90; P0.001) was demonstrated between in vitro BBB permeability and in vivo permeability coefficient. In contrast, a poor correlation (r(2) = 0.17) was obtained between Caco-2 data and in vivo human brain penetration. This study highlights the potential of this in vitro cell-based human BBB model in drug discovery and shows that it can be an extremely effective screening tool for CNS programs.

Efflux proteins have been shown to greatly affect the uptake of antiretroviral drugs by cells and to prevent their access to the HIV-1 replication site. The active efflux of these drugs might produce subtherapeutic drug levels and favor resistant viral strains and the emergence of sanctuary sites. This study has been performed to investigate whether emtricitabine (FTC) is a substrate and/or inhibitor of MRP1 in human peripheral blood mononuclear cells (PBMCs, HIV-1 target site). Moreover, we have reported the impact of FTC combined with protease inhibitors (PIs) (ritonavir, atazanavir, lopinavir) on Pgp and MRP1 expression and function, and on PI accumulation. Following a 72-h incubation with antiretroviral regimen, Pgp and MRP1 expression and function were assessed on lymphocytes; and intracellular drug concentrations were measured by LC–MS/MS. FTC concentrations were determined following incubation with or without specific efflux proteins inhibitors. FTC inhibitor properties were measured using 2 different MRP substrates. Quantitative real-time PCR showed that PBMCs express high levels of both Pgp and MRP1 mRNA copy number whereas MRP2 and MRP3 were not detectable. Our findings indicate a decrease in MRP1 function after exposure to FTC. MK571 (specific MRP inhibitor) significantly increases FTC accumulation in PBMCs. FTC increases intracellular calcein and [ 3 H]-vincristine accumulation. Emtricitabine has both inhibitor and substrate characteristics with MRP1 in PBMCs in vitro, and does not interact with PI accumulation.

Efflux pumps, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP) have been shown to extrude HIV protease inhibitors from cells. These transporters are present on many barrier sites such as the blood-brain barrier (BBB) and on many circulating cells such as lymphocytes, and could reduce protease inhibitor concentration in sanctuary or HIV-1 target sites. This study compares the potential of the antiretroviral drug atazanavir to modulate P-gp and MRP expression and function in total lymphocytes and in human fetal brain endothelial cells (HBMECs). We address the question of atazanavir transport across the human BBB. Following incubation with atazanavir, P-gp and MRP1 expression was determined by direct immunofluorescence. Transporter function was assessed by measuring fluorescent dye efflux, either with or without specific inhibitors. Atazanavir substrate properties were determined by transport quantification through a validated in vitro human BBB model. Our results show that in contrast to HBMECs, in lymphocytes, atazanavir has no effect on MRP1 and P-gp expression. However, there were overall changes in P-gp function increasing its activity in lymphocytes and HBMECs. Using the in vitro human BBB model, we confirm the interaction of atazanavir with P-gp, MRPs, and BCRP in preventing its passage across this barrier and thus its entry into the central nervous system.

Aims It is a huge challenge to understand the blood–brain barrier (BBB), which is a key element in neuroinflammation associated with many brain diseases. The BBB also regulates the passage of xenobiotics into the central nervous system (CNS), and therefore influences drug efficacy. This may be due to the presence of ATP binding cassette transporters such as P-glycoprotein (Pgp) on the BBB, which are efflux pumps known to transport many drugs. The peptide endothelin 1 (ET-1) is involved in different kinds of CNS diseases and neuroinflammation, and is known to modulate Pgp transport activity. Although there are data from animal models, data from human models are scarce. We evaluated Pgp expression and transport activity in adult human brain microvascular endothelial cells (HBMECs) when exposing an adult human in vitro BBB model to ET-1. Methods Adult HBMECs were cocultured with human adult glial cells on a TranswellsR to mimic blood and CNS compartments. These human in vitro BBBs were exposed for 24 h to 100 nM and 10 nM ET-1. Pgp expression was assessed by flow cytometry and its transport activity by measuring radiolabelled digoxin passage. Results After exposure to ET-1, flow cytometry showed no shift of fluorescence intensity for a Pgp specific antibody. The passage of digoxin increased with a significant decrease of Q ratio for 10 nM ET-1. Conclusion Our results show that ET-1 has no effect on Pgp expression of adult HBMECs, but does modulate Pgp transport activity.

P-glycoprotein transports several compounds including protease inhibitors, actually used in the clinical management of HIV-1 infection. Since P-glycoprotein is expressed in placental trophoblasts, its efflux activity could interfere with placental transfer of antiretrovirals. The purpose of this study was to investigate the expression of P-gp-encoding MDR1 gene and P-gp itself in full-term placentas from uninfected (n=35) and HIV-1 infected women (n=24). MDR1 transcripts were quantified by real-time PCR using relative (MDR1 normalized upon 28S levels) and absolute (copy number) determinations. P-glycoprotein localization and expression were evaluated by immunohistochemistry and western blot analysis, respectively. Relative or absolute PCR quantification showed a significant 3.3-fold (p

The blood-brain barrier (BBB) permeabilities of 11 compounds were measured both in vitro with a newly developed coculture-based model of human BBB and in vivo with positron emission tomography (PET). The 11 compounds were fluoropyridinyl derivatives labeled with the positron-emitter fluorine-18, [(18)F]F-A-85380 [2-[(18)F]fluoro-3-[2(S)-2 azetidinylmethoxy]pyridine], and 10 selected N-substituted-azetidinyl and pyrrolidinyl closely related [(18)F]fluoropyridinyl derivatives (including [N'-aromatic/aliphatic]-thioureas, -ureas, and -amides). The in vitro BBB model, a new coculture system of primary human brain endothelial cells and astrocytes, was used to measure the permeability coefficient for each compound. Dynamic PET studies were performed in rats with the same compounds, and a two-compartment model analysis was used to calculate their in vivo permeability coefficients. The 11 derivatives differed in their degree of BBB passage and transport mechanism. The analysis of PET data showed a significant cerebral uptake for six derivatives, for which the in vitro evaluation indicated active influx or free diffusion. Five derivatives displayed low in vivo cerebral uptake, in agreement with the observation of an in vitro active efflux. Overall, there was a remarkable correlation between the in vitro and in vivo permeability coefficients (r = 0.99). This double study proves a close correlationship between the assessment of the BBB passage in vitro and in vivo. The in vitro model of human BBB offers the possibility of subtle discrimination of various BBB permeability degrees and transport mechanisms. Conversely, small animal PET imaging appears suitable to screen directly in vivo brain targeting of drugs or radiopharmaceutical candidates.

HIV protease inhibitors (HPIs) have limited penetration into the brain. This poor transport through the blood-brain barrier is mainly due to active efflux by proteins such as P-glycoprotein (P-gp) preventing drugs from clearing the brain of the virus. The present paper focuses on cerebral uptake of HPIs and interactions between HPIs and efflux proteins, either as substrates or modulators. Most of the studies described HPIs as P-gp substrates. Studies are more controversial when investigating HPIs as inhibitors of P-gp. HPIs seem to be able to inhibit efflux proteins of in vitro cell models but with limited consequences in vivo. Moreover, after repeated administrations of HPIs, most of them are also able to induce the expression and functionality of P-gp. For these reasons, certain combinations of HPIs may not efficiently increase brain uptake of HPIs as would combinations of more potent efflux inhibitors.

Objectives To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. Design: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. Methods P-gp and MRP expression in response to long-term zidovudine (3′-azido-3′-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. Results Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. Conclusion AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

The growing array of in vitro models of the blood-brain barrier (BBB) which have been used makes it difficult to draw firm conclusions concerning the BBB penetration of HIV-1 protease inhibitors. What is needed is a combined in vivo and in vitro study on biological models that mimic as closely as possible the normal human BBB, to establish whether and how indinavir crosses the BBB. We developed a new human BBB model using primary endothelial cells and astrocytes. The biological relevance of this model was checked with respect on the one hand, to the close relationship between the log of drug permeability coefficient normalized to molecular weight and the log of the 1-octanol/water partition coefficient, and on the other hand to the functional P-glycoprotein (P-gp) expression. We employed this model to perform transport studies with indinavir and showed that the rate of in vitro indinavir transport from the basal to apical compartment was higher than the rate of apical to basal transport. Pretreatment of the BBB model with the P-gp inhibitor, quinidine, significantly increased apical to basal transport. Intracellular indinavir accumulation was increased in BBB as a result of inhibition of active transport. These data were correlated with the indinavir-mediated P-gp ATPase modulation showing that indinavir specifically interacted with a binding site on P-gp. Moreover, the activation of P-gp ATPase by indinavir was inhibited by quinidine. In addition, the in vivo brain to plasma concentration ratio of indinavir into mice showed that indinavir concentration was up to five times higher in the brain of mdr1a(-/-) mice than in the brain of mdr1a(+/+) mice. All these results confirm the role of P-gp in preventing the passage of indinavir across BBB and thus its entry into the central nervous system (CNS). Our human BBB model represents a useful tool for the evaluation of drug penetration into the CNS.

Background: Creatine transporter (CT) deficiency is characterized by mutations in the gene encoding CT, leading to impaired transport of creatine at the cell membrane. Patients with this disease would thus benefit from replenishment of creatine inside the brain cells. Aim: We report a therapeutic strategy based on the use of dodecyl creatine ester incorporated into lipid nanocapsules (LNCs). Materials & methods: The dodecyl creatine ester was incorporated in the shells of LNCs using Transcutol® (Gattefossé SAS, Saint-Priest, France). The interactions of dodecyl creatine ester encapsulated in LNCs with an in vitro cell-based blood–brain barrier model was studied. The entry of the dodecyl creatine ester encapsulated in LNCs and the conversion of dodecyl creatine ester to creatine in the cells were also studied in the pathological context of CT deficiency. Results & discussion: We showed that these LNCs can cross the blood–brain barrier and enter brain endothelial cells. In human fibroblasts lacking functional CT, all or part of the dodecyl creatine ester was released from the LNCs and biotransformed to creatine, thus indicating the value of this strategy in this therapeutic context. Original submitted 2 April 2013; Revised submitted 28 October 2013

International audience; BACKGROUND: TiO2 particles are commonly used as dietary supplements and may contain up to 36% of nano-sized particles (TiO2-NPs). Still impact and translocation of NPs through the gut epithelium is poorly documented. RESULTS: We show that, in vivo and ex vivo, agglomerates of TiO2-NPs cross both the regular ileum epithelium and the follicle-associated epithelium (FAE) and alter the paracellular permeability of the ileum and colon epithelia. In vitro, they accumulate in M-cells and mucus-secreting cells, much less in enterocytes. They do not cause overt cytotoxicity or apoptosis. They translocate through a model of FAE only, but induce tight junctions remodeling in the regular ileum epithelium, which is a sign of integrity alteration and suggests paracellular passage of NPs. Finally we prove that TiO2-NPs do not dissolve when sequestered up to 24 h in gut cells. CONCLUSIONS: Taken together these data prove that TiO2-NPs would possibly translocate through both the regular epithelium lining the ileum and through Peyer's patches, would induce epithelium impairment, and would persist in gut cells where they would possibly induce chronic damage.

Further evidence suggests that cell adhesion molecules (CAMs) expressed on the surface of human immunodeficiency virus type 1 (HIV-1)-infected cells are regulated during lentiviral infection. To address this hypothesis we have investigated the kinetic pattern of CAM expression at the surface of HIV-1Ba.L-infected human monocytes during the first 72 hr of infection. A significantly lower expression of CD18 and CD54 as well as a decrease in CD44 expression level were observed at the surface of infected monocytes when compared with mock-infected cultures. No modification of CD11a, CD11b, CD11c, CD58, and CD62L expression was detected. Except for CD18, the expression of which at the cell surface is decreased, no modification of CD44 and CD54 expression was observed after heat-inactivated HIV-1 treatment of monocytes. Investigation of soluble forms of CAMs (sCAMs) and cytokine production in the culture supernatants of infected monocytes showed a peak of sCD44, TNF-alpha, IL-1beta, and IL-6 release between 2 and 24 hr after infection. Treatment of monocytes with monoclonal antibodies (MAbs) against CAMs showed that engagement of some CAMs may trigger TNF-alpha and IL-1beta production. In addition, pretreatment of infected monocytes with a TNF-alpha synthesis inhibitor, RP 55778, or with MAbs directed against IL-1beta, confirmed the role of TNF-alpha and IL-1beta in the regulation of CD18, CD44, and CD54 expression.

Clinical and biological features indicate that a dysregulation of microbicidal activity occurs in the cells of mononuclear phagocytic lineage of HIV-1-infected patients. Thus, the regulation of MnSOD gene expression has been investigated during the 10 h following in vitro HIV macrophage infection. As previously reported, in HIV-1 LAI-infected macrophages a high expression of the MnSOD gene is observed 2 and 4 h after infection. These results are confirmed when cells are infected with three macrophage-tropic strains HIV-1 DAS, PAR and Bal. Moreover, the detection of the MnSOD gene expression in the macrophage cultures is associated with the cellular tropism of the viral strains used. The binding of recombinant GP160 by itself is not sufficient to induce MnSOD expression. In fact, the same MnSOD gene induction was obtained with the heat inactivated viral isolates, indicating that these phenomena are due to the viral entry. On the other hand, phagocytosis of latex beads triggers a high expression of the MnSOD gene in macrophages, showing that phagocytosis of HIV may be sufficient to induce the expression of that gene. Taken together, these results indicate that the MnSOD gene expression observed within 10 h following infection of macrophages is mainly related to membrane biophysical unspecific modifications.

Human monocytes/macrophages, which express Fc receptors for IgG are involved in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis. These receptors are known to mediate numerous immunological functions including cell-mediated killing and possibly targeting of HIV to the lysophagosome monocyte-derived macrophage (MDM) entry route for virus neutralization. To study both activities in HIV-1 infection, MDM Fc gamma RI was specifically selected using bispecific antibody (Bs-Ab) containing whole human monoclonal antibody against gp41 and the Fab' fragment of murine anti-Fc gamma RI 22.2 antibody. Bs-Ab was found to mediate potent antibody-dependent cellular cytotoxicity and virus neutralization.

Monocyte-derived macrophages (MDM) were demonstrated to be susceptible to productive infection by the monocytotropic human immunodeficiency virus type 1 (HIV-1) strain HIV-1/Ba-L and by three primary HIV-1 isolates, HIV-1/DAS, HIV-1/PAR and HIV-1/THI. Production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-1 beta was monitored between days 3 and 26 after MDM infection. TNF-alpha and IL-6 were detected in cell culture supernatants from days 16 to 21 following HIV-1/DAS, HIV-1/PAR and HIV-1/Ba-L infection, at the time of high viral replication. IL-1 beta was not found at the same time points. TNF-alpha mRNA expression occurred around the peak of both TNF-alpha levels and supernatant RT activities. In HIV-1/THI-infected macrophage cultures no endogenously produced TNF-alpha was observed, despite high levels of HIV-1 in MDM. This result demonstrates that a primary isolate may replicate independently of TNF-alpha in MDM. To investigate the relationship between TNF-alpha and viral replication we used a TNF-alpha synthesis inhibitor, RP 55778. Treatment throughout the course of cell culture resulted in a significant decrease in both TNF-alpha levels and viral production in HIV-1/DAS-, HIV-1/PAR- and HIV-1/Ba-L-infected MDM cultures. This phenomenon is reversed by adding recombinant human TNF-alpha to the RP 55778-treated cell cultures from day 14 post-infection. No effect of RP 55778 was observed in MDM cultures infected with the primary isolate HIV-1/THI, whose replication is independent of TNF-alpha production and therefore remained unchanged after RP 55778 treatment. We conclude that the clinical value of such a drug is directly dependent on the ability of the HIV-1 strains involved to induce TNF-alpha production at the time of viral replication.

Human monocyte-derived macrophages (MDM) were infected with the viral strain HIV1/Ba-L and with the clinical isolates HIV1/DAS and HIV1/PAR. Kinetics of tumour necrosis factor alpha (TNF alpha) and interleukin-6 (IL6) production were investigated for 28 days after infection. At the early stages of infection we observed significant TNF alpha and IL6 secretion 2 to 10 h after infection, whatever the viral strain we used. During the late events of MDM infection, TNF alpha and IL6 were detected over 16 to 21 days following HIV1 infection, at the time of high viral replication. Pretreatment of MDM with a TNF alpha synthesis inhibitor, RP 55778, 4 h prior to HIV infection induced a modified cytokine pattern during the first ten hours of infection: TNF alpha production was totally inhibited despite comparable amounts of IL6. At the late phases of the cell culture, a decrease in magnitude of both viral and cytokine production as well as a delay in the appearance of reverse transcriptase activity and cytokine secretion peaks were observed in RP-55778-pretreated and HIV1-infected MDM cultures. Similar results were obtained after pretreatment of HIV1/DAS-infected MDM cultures with an anti-TNF alpha monoclonal antibody.

Brain penetration is characterized by its extent and rate and is influenced by drug physicochemical properties, plasma exposure, plasma and brain protein binding and BBB permeability. This raises questions related to physiology, interspecies differences and in vitro/in vivo extrapolation. We herein discuss the use of in vitro human and animal BBB model as a tool to improve CNS compound selection. These cell-based BBB models are characterized by low paracellular permeation, well-developed tight junctions and functional efflux transporters. A study of twenty drugs shows similar compound ranking between rat and human models although with a 2-fold higher permeability in rat. cLogP5, PSA120 Å, MW450 were confirmed as essential for CNS drugs. An in vitro/in vivo correlation in rat (R² = 0.67; P = 2 × 10⁻⁴) was highlighted when in vitro permeability and efflux were considered together with plasma exposure and free fraction. The cell-based BBB model is suitable to optimize CNS-drug selection, to study interspecies differences and then to support human brain exposure prediction.

Monocyte/macrophage infection by human immunodeficiency virus type 1 (HIV1) was studied for its effects on the production of tumour necrosis factor alpha (TNF alpha) and the expression of the manganese superoxide dismutase (MnSOD) gene. For this purpose, human peripheral blood monocytes were obtained from healthy HIV1-seronegative donors by centrifugal elutriation and infected with either the HIV1/LAV1 strain or with the primary HIV1/DAS isolate. The results showed that (1) HIV1/LAV1-infected macrophages did not produce any biologically detectable TNF alpha during the few hours following lentiviral infection, despite rises in the TNF alpha mRNA level; (2) MnSOD gene transcription in the macrophages increased, as measured 2 and 4 h after infection; (3) the level of the MnSOD gene expression declined during the late phases of lentiviral infection, but TNF alpha synthesis and gene expression rose; and (4) bispecific antibody comprised of anti-Fc gamma RI (anti-CD64) and anti-gp41 monoclonal antibodies inhibited the in vitro infection of monocyte-derived macrophages by HIV1/DAS.

The advent of early absorption, distribution, metabolism, excretion, and toxicity (ADMET) screening has increased the attrition rate of weak drug candidates early in the drug-discovery process, and decreased the proportion of compounds failing in clinical trials for ADMET reasons. This paper reviews the history of ADMET screening and its place in pharmaceutical development, and central nervous system drug discovery in particular. Assays that have been developed in response to specific needs and improvements in technology that result in higher throughput and greater accuracy of prediction of human mechanisms of absorption and toxicity are discussed. The paper concludes with the authors' forecast of new models that will better predict human efficacy and toxicity.

Efflux proteins have been shown to greatly affect the uptake of antiretroviral drugs by cells and to hamper their access to the human immunodeficiency virus type 1 replication site. This study evaluated the factors that may lead to drug-drug interactions between emtricitabine (FTC), tenofovir (TFV), and efavirenz (EFV), including the modulation of efflux transporter expression and function. Peripheral blood mononuclear cells from healthy volunteers were used to determine whether or not an interaction between antiretroviral drugs and target cells occurred in any combination of FTC, TFV, EFV, FTC-TFV, TFV-EFV, or FTC-TFV-EFV. Following 20 h of treatment, intracellular drug concentrations were measured by liquid chromatography-tandem mass spectrometry. Efflux transporter functionality and inhibitor drug properties were assessed by measuring fluorescent dye efflux. ABCB1 (P-glycoprotein), ABCC 1 to 6 (multidrug resistance-associated protein), and OAT (organic anion transporter) expression in response to the treatments was quantified by semiquantitative real-time PCR. Cells treated with a double combination (FTC-TFV or TFV-EFV) or the triple combination (FTC-TFV-EFV) produced higher FTC and TFV intracellular concentrations than cells treated with FTC or TFV alone. However, no change in the EFV intracellular concentration was observed. FTC tended to induceabcc5mRNA expression and EFV tended to induceabcc1andabcc6mRNA expression, whereas TFV tended to reducemdr1,abcc1,abcc5, andabcc6mRNA expression. Under these conditions, a decrease in the functionality of ABCC was observed, and this decrease was associated with the direct inhibitory actions of these drugs. This in vitro study reveals a benefit of the combination FTC-TFV-EFV in terms of the intracellular FTC and TFV concentrations and highlights the pharmacological mechanisms that lead to this effect.

Most attempts to produce a vaccine against HIV-1 infection are utilizing envelope protein components. Hypothetically such vaccine candidates could stimulate production of antibodies that enhance HIV-1 infection via the macrophage route of entry and, consequently, cannot be detected in the conventional neutralization assay. To study this hypothesis we report an assay designed to evaluate the protective/enhancing activity of serum from seropositive immunized or infected individuals. Highly purified activated FcR-bearing monocytes-macrophages were infected with HIV-1 in the presence of the sera, then washed and cocultured with activated peripheral blood mononuclear cells (PBMC) from a normal donor. Productive viral infection, as evaluated by p24 antigen semiquantitative assay in the culture supernatants, allow evaluation of protective/enhancing activity of the sera. The data clearly show that protective rather than enhancing activity is present in the serum of env protein-immunized individuals.

We present a simple chromatographic method to detect and quantify protease inhibitors (PI), metabolites and non-nucleoside reverse transcriptase inhibitors (NNRTIs) in human plasma of HIV-1 infected patients and in peripheral blood mononuclear cells (PBMCs) using either liquid chromatography coupled with ultraviolet (LC–UV) or liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). A solid–liquid extraction was carried out on 500 μl of plasma as pre-treatment. Calibration curve ranges were from 50 (100) to 5000 ng/ml (indinavir). PBMC pellets from 7 ml of blood were lysed with methanol/tris with a calibration curve ranging from 0.25 to 250 ng/pellet. Simple modifications in the mobile phase composition (slight increase of ammonium acetate concentration and addition of methanol for LC–UV) easily linked the two analytical systems.

The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Evidence suggests that endothelin-1 (ET-1) plays an essential role in brain inflammation. However, whether ET-1 contributes directly to blood-brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co-cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET-1 by BMVECs upon stimulation with tumour necrosis factor (TNF)-alpha, which plays an essential role in the induction and synthesis of ET-1 during systemic inflammatory responses. Increased ET-1 mRNA was detected in the human BMVECs 24 h after TNF-alpha treatment. This was correlated with an increase in ET-1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF-alpha and ET-1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF-alpha was partially prevented by antibody neutralization of ET-1 and completely by monoclonal antibody against IL-1beta. Concomitantly, TNF-alpha induced IL-1beta mRNA expression by astrocytes in co-culture and this effect was partially prevented by ET-1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET-1 for 24 h induced IL-1beta mRNA synthesis and IL-1beta protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET-1, TNF-alpha and IL-1beta between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.

Evidence suggests that the HIV-1 envelope glycoproteins play a role in the central nervous system (CNS) complications of AIDS. Endothelin-1 (ET-1) has also been implicated in brain injury and the progression of the AIDS dementia complex (ADC). Here, we used a real-time reverse transcription polymerase chain reaction assay and an immunometric assay to show that in vitro model of the blood-brain barrier (BBB) consisting of a monolayer co-culture of astrocytes and human brain microvascular endothelial cells (A-HBMEC) increased its expression of ET-1 mRNA and secretion of ET-1 peptide when infected with HIV-1. The enhanced expression of ET-1 occurred independently of viral replication as it was also induced by the viral glycoprotein coat HIV-1g120SF. These results show that one mechanism by which HIV-1 might affect the CNS is by inducing release of ET-1 by the BBB.

Antiretroviral effects of a new class of interferon (IFN), IFN-tau, were compared with those of IFN-alpha in primary peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs), infected in vitro by human immunodeficiency viruses type 1, HIV-1/LAI, and HIV-1/DAS isolates, respectively. Cells were treated with recombinant IFN 24 h before or after HIV infection and then continuously exposed. Viral replication was monitored twice a week by quantifying the reverse transcriptase activity in cell culture supernatants. Integrated proviral DNA was monitored 24 h after infection in IFN-tau-pretreated MDMs, using specific gag gene amplification by the polymerase chain reaction. IFN-tau inhibited HIV-1 replication in both PBLs and MDMs as well as in peripheral blood mononuclear cells (PBMCs). IFN-tau was 35-fold more potent than IFN-alpha in PBLs and 100-fold more potent in MDMs. Differences were observed in the amount of integrated proviral DNA between untreated and 10 IU/ml IFN-tau-treated HIV-infected MDMs. IFN-tau exhibits significant anti-HIV activity in comparison to IFN-alpha, and like other IFNs, it seems to interact with several steps of HIV replication cycle.

Human monocytes/macrophages play a major role in pathogenesis of human immunodeficiency virus (HIV) infection. These cells have been suspected of acting as a reservoir for the virus and are important in viral dissemination and persistence in infected individual. Furthermore, several biologic and clinical features indicate that monocytes/macrophages from HIV-1-seropositive patients have characteristics of an activation status, including the ability to secrete high levels of cytokines. Dysregulation of the cytokine network may influence the level and the consequences of viral replication in infected monocytes/macrophages. Therefore, the development of virus-specific agents that may interfere with viral replication could help to slow down the fatal course of HIV infection. In this article, we try to further quantify the early and late kinetic patterns of the cytokine network during HIV-1 macrophage infection and report the biologic effects of virus-specific bispecific antibody (MDX-240) in HIV-1 macrophage infection.

A case of HIV infection clearance in a perinatally infected infant has been recently reported. We report here on the molecular, biological and clinical features of such virus clearance in 12 children.We performed a retrospective analysis of the diagnosis in our 6-year cohort of 188 children born to HIV-seropositive mothers. HIV-1 was detected by coculture of infant peripheral blood mononuclear cells (PBMC) with cord blood cells, direct culture of infant cells, and DNA polymerase chain reaction (PCR). The children were diagnosed three times during the first 3 months of life and then followed up over a postnatal period of 18-36 months.The 12 reverted children had at least two positive PCR in at least two amplified regions. Among them, six were tested positive in culture/coculture assay, and five were treated long-term with zidovudine. Thus, seven out of 12 reversions cannot be attributed to antiretroviral therapy. All the virological results became negative during the first year of life, and serology lowered to negative values between 9 and 23 months. We could not find any correlation between either neutralizing or antibody-dependent cellular cytotoxicity-mediating antibodies and HIV clearance.In our cohort, we showed that an unexpected number of children born to HIV-seropositive mothers (6.7%) cleared HIV infection during the first year of life, and subsequently became seronegative. Interestingly, most of these children exhibited unspecified clinical signs during the first months of life. Five of these children were tested positive only by PCR, which suggests a low virus load and could, at least partly, explain spontaneous clearance. However, 4 years later, among the seven remaining infants, two seronegative children presented recurrent hepatosplenomegaly, which may indicate the presence of hidden virus not detectable by peripheral blood testing.

SUMMARYBased on what is known about the biology of HIV-1 vertical transmission, the HIV burden of the mother, maternal immune factors and the integrity of the placental barrier are likely to play major roles. We therefore sought to determine whether the presence of antibodies in sera from 47 HI V-1- infected mothers, including 30 non-transmitting and 17 transmitting mothers, affected the risk of HIV-1 transmission to infants. Our findings showed no significant correlation between the capacity of antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and their capacity to induce protection of the child from HIV-I infection (p=0.14). Furthermore, no correlation was found between the capacity of maternal antibodies to neutralize in vitro lymphocyte or macrophage heterologous viral infection and the occurrence of in vivo HIV-1 infection in the infant. Sera recovered from five of 12 transmitting mothers and from five of 11 non-transmitting mothers were compared in their capacity to neutralize the viruses drawn from the same individuals. Four out of five maternal isolates from transmitting mothers and all maternal isolates from non-transmitting mothers were sensitive to enhancement of infection mediated by the maternal serum.

The macrophage-secreted 92-kDa type IV collagenase and metalloproteinases play a critical role in cell microenvironment regulation and cell movement. HIV infection of macrophages might be capable of deregulating the expression of these gelatinases. Hence, human monocyte-derived-macrophages were infected by lymphotropic HIV-1/Lai and monocytropic HIV-1/DAS isolates. Gelatinase activity and gelatinase and inhibitor (TIMP, α2M) biosyntheses were evaluated in supernatants and cellular extracts. Our data suggest that HIV infection facilitates gelatinase secretion and intracellular inhibitor retention. These argue for the increase of free proteinase that could degrade barriers, which would permit cell movement and viral dissemination into tissues.

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.

Human monocyte-derived macrophages that express the CD4 molecule and the Fc receptor for IgG (Fc gamma R) play a major role in the pathogenesis of human immunodeficiency virus (HIV) infection. To explore this possibility further, human monoclonal antibody to glycoprotein 41 (gp41) was produced, and a heterobifunctional antibody composed of F(ab') x F(ab')2 fragments of monoclonal anti-gp41 and anti-Fc gamma RI 22.2 were constructed. Both antibodies were analyzed for neutralizing effects, and the role of the CD4 molecule in HIV infection was studied with human monocyte-derived macrophages. The bispecific antibody exhibited strong neutralizing properties, in contrast to the monoclonal anti-gp41 antibody. Moreover, in the presence of monoclonal anti-Leu-3a antibody, viral production was completely inhibited. These findings demonstrate the necessity of the CD4 molecule in HIV infection of human macrophages and emphasize the usefulness of such heterobifunctional antibody directed to virus and monocyte-derived macrophage Fc receptors in prevention of HIV infection.

This study compares the antitumoral properties of isolated circulating human blood monocytes (Mo) and of mature macrophages (MO) obtained by 7 days differentiation of Mo or isolated from alveolar washing. These cells were activated to cytotoxicity in the presence of recombinant human interferon-gamma (rHuIFN-gamma). This antitumoral effect was measured at a low (1/1) effector/target ratio without pretreatment of the tumor cells. Activated Mo released tumor necrosis factor-alpha (TNF-alpha) in the culture medium where their antitumoral activity could be totally neutralized by specific anti-rHuTNF-alpha antibodies. In contrast, blood monocytes derived macrophages differentiated and activated in vitro expressed TNF-alpha on their membrane where it could be labelled and partially neutralized by anti-rHuTNF-alpha antibodies. Direct effector/target contact was required for the activity of macrophages differentiated in culture or collected from the lung cavity of healthy subjects. When these macrophages were obtained from infected patients or subjected to LPS treatment, they directly released cytotoxic amounts of TNF in the extracellular fluid after activation with IFN-gamma. Monocytes act mainly by soluble mediators (TNF-alpha being a key factor), while differentiated macrophages in the absence of endotoxin act by close cell to cell contact involving the lytic action of membranous TNF-alpha as well as some release of soluble TNF-alpha. We also present evidences (based on the use of various protease inhibitors) that the role of proteases is much less crucial in the cytotoxic action of monocytes and macrophages.

International audience; Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aβ), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aβ accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-β Forty-Two Inducer named Aftin-5. Secreted, soluble Aβ fragment concentrations were analyzed in MB-conditioned media. An increase in the Aβ 42 fragment secretion was observed as was an increased Aβ 42 /Aβ 40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aβ accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aβ concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aβ oligomers and their target, the cellular prion protein (PrP C), support the possibility of using MBs to further understand the patho-physiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.